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Department of Public Health and Family Medicine, Tufts University School of Medicine, Boston, University of Massachusetts Medical School, Worcester, Harvard Medical School, New England Regional Primate Research Center, Southborough
Department of Clinical Sciences, Tufts University School of Veterinary Medicine, North Grafton, Massachusetts
Background.
We investigated the impact that micronutrient supplementation has on the progression of simian acquired immunodeficiency syndrome (SAIDS).
Methods.
Twenty-four simian immunodeficiency virusinfected juvenile male rhesus macaques were randomized into 2 groups. One group was given certified chow, and the other group was given chow and a supplement that contained 23 times the estimated nutritional requirement of micronutrients. Virological, immunological, and body composition measurements were taken every 4 weeks for 120 weeks.
Results.
There was no difference between groups in weight gain, body mass index (BMI), crown-heel length, waist circumference, total tissue mass, lean mass, bone mineral content, or bone mineral density. The rhesus macaques on the supplemented diet had a higher death rate (hazard ratio, 2.39; P < .001) than those on the nonsupplemented diet; death in both groups was associated with a higher viral load set point during the early phase of infection. Additionally, higher body weight, BMI, crown-rump length, and lower viral load set point were protective from death in both groups.
Conclusions.
Micronutrient supplementation did not significantly alter the progression of SAIDS with respect to changes in body composition and immunological characteristics. A significantly higher rate of death was observed in rhesus macaques on the supplemented diet.
The simian immunodeficiency virus (SIV)infected rhesus macaque model has been useful in the exploration of facets of HIV infection and AIDS that are difficult to study in humans. During the era before the use of highly active antiretroviral therapy (HAART), patients infected with HIV were shown to have levels of selected micronutrients (selenium, niacin, and vitamins A, B1, B2, B6, B12, and E) that were lower than desirable [14]. These micronutrient deficiencies were associated with more-rapid progression of HIV disease than was observed in micronutrient-replete patients. Because compelling data suggest that deficiencies of select micronutrients may have an adverse effect on immune function, it is important to better understand the interaction between micronutrients and HIV disease progression. In addition, there is widespread use of micronutrient supplementationfrom physiological replacement to supraphysiological (pharmacological) dosesin patients with HIV disease, but there is no clear understanding of the impact that this supplementation has [58]. To date, only 1 study of the role that micronutrients play in simian acquired immunodeficiency syndrome (SAIDS) in the SIV-infected rhesus macaque model has been published [9]. In that study, SIV-infected rhesus macaques showed a decrease in blood selenium levels with the development of SAIDS. As was shown by multiple measurements taken between the time of infection and death, there was a significant decline in blood selenium levels in the 9 female rhesus macaques studied (P < .01).
Two previous studies from our group have used the SIV-infected rhesus macaque model to investigate the effects that SIV infection has on growth and body composition [10, 11]. In the first study, we found that the SIV-infected rhesus macaque model was valid in the exploration of the impact that SIV infection has on growth in juvenile male rhesus macaques, because SIV infection caused failure to thrive in this model; the magnitude of this effect was related to the severity of infection [10]. In the second study, also using juvenile male rhesus macaques, we found that SIV infection caused a 3-phase alteration in body composition that is similar to that observed in HIV-infected humans during the pre-HAART era [11, 1217]. The first phase was an initial loss of body weight, predominantly from fat tissue, which was followed by a second phase of compensatory growth, albeit at a reduced rate. The final phase, which occurred just before death, was a loss from all body compartments. The study of micronutrients in humans with HIV infection is confounded by variations in dietary intake and micronutrient supplementation, differences in intestinal function and absorption, variations in the severity of HIV disease, and the complexities of using multiple therapies. Use of the SIV-infected rhesus macaque model can circumvent these issues. In this study, we used SIV-infected rhesus macaques to compare outcomes in rhesus macaques receiving a standard micronutrient supplement (that had 23 times the estimated nutritional requirement of micronutrients) to outcomes in a control group of matched rhesus macaques receiving adequate levels of micronutrients in their regular diet.
MATERIALS AND METHODS
Rhesus macaques.
Twenty-four juvenile male rhesus macaques were studied. All rhesus macaques were injected intravenously (week 0) with SIV mac239 (50 ng of p27 viral antigen equivalent). The rhesus macaques were then randomized into 2 groups. One group (n = 12), with a mean age of 68.6 weeks, was given the nonsupplemented control diet. The other group (n = 12), with a mean age of 67.0 weeks, was given the supplemented diet (table 1). The baseline characteristics of the 24 rhesus macaques are given in table 1. There was no statistically significant difference between the groups in baseline characteristics, except in age at time of inoculation (P = .04). Although age was different, the magnitude of the difference was small (1.6 weeks). There was also no statistical difference between the groups in baseline immunological parameterssuch as CD4+ and CD8+ cell and lymphocyte countsthat were measured in this study.
All rhesus macaques were housed at the New England Regional Primate Research Center (NERPRC) and were maintained in accordance with the Guide for the Care and Use of Laboratory Animals [18]. The facility is accredited by the American Association for the Accreditation of Laboratory Animal Care. Clinical procedures were performed under the direction of a veterinarian, and all possible measures were taken to minimize discomfort in the rhesus macaques. Appropriate anesthesia and analgesics were used and administered under the direction of a veterinarian. Telazol was routinely used during the handling of the rhesus macaques. Euthanasia criteria were developed before the initiation of the study, were approved by the Harvard Medical School Standing Committee on Animals, and were consistent with those used in other SAIDS studies performed at the NERPRC. The criteria were (1) weight loss of >15% in 2 weeks; (2) documented opportunistic infection; (3) persistent anorexia for >3 days without explicable cause; (4) severe, intractable diarrhea; (5) progressive neurological signs of illness; (6) significant cardiac and/or pulmonary signs of illness; and (7) any other serious illness. A veterinarian blinded to the treatment groups performed assessments of the rhesus macaques. If euthanasia was required in the judgment of the veterinarian, it was carried out in accordance with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association.
Diet.
All rhesus macaques were maintained on a certified chow in biscuit form (Purina no. 5048; provided by Purina Mills), were fed twice per day, and were allowed to eat as much as desired. The rhesus macaques were acclimated to the diet before infection. This diet was selected to guarantee a nutrient variability of 5% from batch to batch and provide the necessary recommended nutrient requirements for rhesus macaques. Food intake was monitored and recorded daily, and loss due to discarded biscuits was taken into consideration. A biscuit designed to provide 23 times the estimated nutritional requirement of micronutrients was given daily to the rhesus macaques in the supplemented group (table 2 shows the levels of micronutrients in the nonsupplemented diet and in the supplemented diet). A placebo biscuit made of filler carbohydrate was given daily to the rhesus macaques in the nonsupplemented group. Both biscuits were orange flavored, to ensure consumption by the rhesus macaques. Micronutrient increases in the supplemented diet were based on the usual intake for 23-year-old rhesus macaques at 3 kg of body weight. The rhesus macaques in the supplemented group were also given monthly intramuscular injections of 200 g of vitamin B12. The supplemented diet, which incorporated daily micronutrient intakes that were 23 times the estimated nutritional requirement, was selected to simulate levels shown to be protective for HIV-infected humans [4, 2023].
Body composition.
Body weight was assessed every 4 weeks using a calibrated scale that measured to the nearest 0.01 g. The following somatometric measurements were taken: crown-heel length (from the top of the unflexed head to the bottom of the heel), crown-rump length (from the top of the unflexed head to the junction of the rump-tail groove), knee height (with the leg flexed, from the top of the knee to the bottom of the heel), upper arm circumference (at the midbiceps level), upper leg circumference (at the midquadriceps level), chest circumference (at the level of the nipples), and abdominal circumference (at the level of the umbilicus). Body mass index was calculated as (body weight in kilograms)/(crown-heel length in centimeters)2.
Measurement of specific body compartments (fat mass, lean mass, bone mineral content, and bone mineral density) was performed by dual energy x-ray absorptiometry (DEXA) using a Lunar Prodigy Bone Densitometer (GE Lunar), and results were analyzed using software developed by the manufacturer specifically for analyzing soft tissue of small primates (version 5.00.211). Changes in the body composition of rhesus macaques on the nonsupplemented diet have been described elsewhere [11].
Viral load and immunophenotyping.
Viral load was determined by reverse-transcription polymerase chain reaction, as described elsewhere [24]. Cell immunophenotyping was performed to determine CD4+ and CD8+ T cell counts and percentages. Antibodies used for immunophenotyping of lymphocytes included anti-CD3 (6G12), anti-CD4 (OKT4), and anti-CD8 (LEU-2a). Cells were incubated with antibodies in the presence of staining media (PBS with 2% mouse serum). After antibody staining, the cells were fixed with fresh 2% paraformaldehyde. Three-color flow-cytometric analysis of the cells was performed using a FACScan and CellQuest software (version 3.2; BD Biosciences). Appropriate isotype controls were used to establish positive and negative gates. To exclude cellular debris, 20,000 events were collected from a live gate.
Statistical analysis.
To compare the difference in responses between SIV-infected rhesus macaques on the nonsupplemented diet and those on the supplemented diet, cross-sectional time series generalized least-squares models with first-order autoregressive disturbances were used. A significant number of SIV-infected rhesus macaques died during the course of the study, and death was correlated with the change in response variables of interest. To account for this dropout, the available observations of SIV-infected rhesus macaques were weighted according to the inverse of the estimated survival probabilities for the SIV-infected rhesus macaques. The survival probabilities were estimated using a Weibull regression model in which age and each response variable were time-dependent covariates. Results were considered to be significant when 2-tailed P < .05. Variance and covariance were estimated using the bootstrap method [25]. To evaluate the effect that dietary supplementation has on survival after infection, a Weibull regression model was used in which dietary supplement, CD4+ cell count, viral load set point value (log10 scale), BMI, and age at infection were predictors. The variance-covariance matrix was estimated using a robust procedure. All computations were performed using Stata SE software (version 8.2; StataCorp).
RESULTS
Effect of infection and supplementation on anthropometrics and body composition.
Both the nonsupplemented and the supplemented group had a nonlinear weight gain over time (P < .001 for the nonsupplemented group; P = .001 for the supplemented group), and the body weight growth rates of the 2 groups were not statistically different (P = .430) (table 3). Although an initial decline in body weight growth rate as a result of SIV infection was observed in both the nonsupplemented and the supplemented group (P < .001 for both groups), the body weight growth rate accelerated over time in both groups. Although the nonsupplemented group showed a more rapid acceleration of weight gain, this was not significant (P = .430). Additionally, although both the nonsupplemented and the supplemented group showed an increase in BMI over time (P < .001 for both groups), there was no significant difference between the 2 groups (P = .883).
Both the nonsupplemented and the supplemented group displayed significant growth in crown-heel length (P < .001 for both groups), waist circumference (P < .001 for the nonsupplemented group; P = .003 for the supplemented group), and crown-rump length (P < .001 for both groups) during the course of the study. However, no significant differences were observed between the nonsupplemented and supplemented groups.
On the basis of DEXA analysis (table 3), the nonsupplemented and supplemented groups displayed significant growth in total tissue mass (P < .001 for both groups), an increase in lean mass (P < .001 for both groups), an increase in bone mineral content (P < .001 for both groups), and an increase in bone mineral density (P < .001 for both groups) during the course of the study. Although measurements in the nonsupplemented group increased at a faster rate in all categories, none of the differences between the groups was significant.
SIV infection caused a loss of fat mass (100 g [95% confidence interval {CI}, 45169 g]; P = .002), a decrease in the percentage of fat mass (4.2% [95% CI, 2.5%6.5%]; P < .001), and an increase in the percentage of lean mass (4.2% [95% CI, 2.4%6.3%]; P < .001) in both groups at 4 weeks after infection. After the initial 4-week postinfection period, lean mass increased over time in both groups (P < .001); however, fat mass remained stable in both groups until 56 weeks after infection and then increased slightly (P < .05). As a result, the percentage of fat mass in both groups continued to decline during the course of the study (P < .01).
Effect of infection and supplementation on serum measurements.
As is presented in table 3, both the nonsupplemented and the supplemented group exhibited a progressive decrease in CD4+ cell counts (P < .001 and P < .025, respectively) and in the percentage of CD4+ cells (P = .006 and P = .005, respectively) over time. The rates of the decreases in CD4+ cell counts and the percentage of CD4+ cells were similar (P = .736 and P = .707, respectively). Although CD8+ cell counts and the percentage of CD8+ cells decreased over time as well, only the decrease in CD8+ cell counts in the nonsupplemented group was statistically significant (P = .001). Weak declining trends in viral load were observed in both the nonsupplemented and the supplemented group (P = .017 and P = .002, respectively), and the trends were similar (P = .901).
Effect of infection and supplementation on death and survival.
Over the course of 120 weeks, 19 of 24 rhesus macaques became moribund from SIV disease progression and were euthanized. Of these rhesus macaques, 10 were in the supplemented group and 9 were in the nonsupplemented group. The supplemented group had a significantly shorter survival time (P < .001) (figure 1). The hazard ratio (HR), as estimated using a Weibull regression model, indicated that the supplemented group had a >2-fold increased risk of death (HR, 2.39 [95% CI, 1.663.44]; P < .001), compared with the nonsupplemented group, after adjustment for age at infection, baseline BMI, baseline CD4+ cell count, log of the viral load set point value, and clustering of experimental groups (table 4).
In both the nonsupplemented and the supplemented group, a lower risk of death was related to higher weight gain (P < .001 and P = .008, respectively), BMI (P = .002 and P < .001, respectively), and crown-rump length (P = .043 and P = .042, respectively) (table 5). Although there were no significant differences in these factors between groups, the protective effect of an increase in body weight (P = .079) and BMI (P = .035) appeared to be stronger in the supplemented group than in the nonsupplemented group. In addition, increases in upper leg circumference (P < .001 for the nonsupplemented group; P < .001 for the supplemented group), abdominal fat thickness (P < .001 for both groups), and thigh fat thickness (P = .001 for the nonsupplemented group; P < .001 for the supplemented group) reduced the risk of death in both groups, but no differences were observed in the magnitude of reduction between the groups. Higher values for lean mass (P < .001 for the nonsupplemented group; P = .004 for the supplemented group), monthly lean mass loss (P > .001 for the nonsupplemented group; P = .014 for the supplemented group) were also related to a lower risk of death. Increases in fat mass offered a significant protective effect in the supplemented group (P = .009) but not in the nonsupplemented group (P = .181). Increase in bone mineral content showed no protective effect for either group (P = .247 for the nonsupplemented group; P = .092 for the supplemented group). Bone mineral content was not related to death in either group (P = .247 for the nonsupplemented group; P = .092 for the supplemented group). However, an increase in bone mineral density was protective in the nonsupplemented group (HR, 0.71 [95% CI, 0.550.92]; P = .009) but not in the supplemented group (HR, 0.97 [95% CI, 0.771.23]; P = .832). The difference between the 2 groups showed a trend toward statistical significance (P = .071).
As is shown in table 5, a higher viral load was associated with a >4-fold increase in death in both the nonsupplemented (HR, 4.22 [95% CI, 1.4012.7]; P = .010) and the supplemented (HR, 4.62 [95% CI, 1.6413.0]; P = .004) group, and there was no difference in HRs between the 2 groups (P = .898). Supplementation appeared to have a significant impact on the effect of the percentage of CD4+ cells on death (P = .012): the supplemented group showed a significant elevation of risk with increased percentage of CD4+ cells (HR, 1.07/1% increase [95% CI, 1.011.14/1% increase]; P = .032), whereas the nonsupplemented group showed an insignificant decrease of risk (HR, 0.97/1% increase [95% CI, 0.931.02/1% increase]; P = .235). There was no association between death and CD4+ cell count, CD8+ cell count and percentage, and lymphocyte count and percentage in either group.
DISCUSSION
Epidemiological and intervention studies of micronutrients have been performed in persons infected with HIV [2023, 26]. Some studies have shown beneficial effects, whereas others have demonstrated deleterious effects, especially with vitamin A supplementation. Fawzi et al. studied HIV-infected pregnant women in Tanzania and found that vitamin A supplementation during gestation and lactation significantly increased the risk of HIV transmission to their children [20]. A multivitamin supplement excluding vitamin A resulted in a small reduction in the transmission of HIV through breast-feeding, but it was not statistically significant.
A more recent study by the same group of researchers analyzed the effect that multivitamin supplementation has on HIV disease progression in the same cohort of 1078 pregnant women in Tanzania [21]. The results showed that 25% of the HIV-infected women who received multivitamins progressed to stage 4 disease or died, compared with 31% of the women in the nonsupplemented group (P = .04). The multivitamin supplementation also resulted in a lower risk of progressing to stage 4 disease, significantly higher CD4+ and CD8+ cell counts, and significantly lower viral loads. It was also noted that addition of vitamin A to the multivitamin regimen reduced some of these beneficial effects, and this finding may be related to what was observed in our SIV study.
Other observations have been made about the effect that serum levels or intake of micronutrients have on HIV infection. It has been reported that 37% of patients with AIDS in the pre-HAART era had vitamin B12 levels below reference values [27]. In a longitudinal 8-year follow-up study of dietary intake and disease progression in patients with AIDS, also in the pre-HAART era, Tang et al. reported that survival was increased in subjects in the highest quartile of niacin and vitamin B1, B2, B6, and B12 intake [22]. The relative HRs were 0.450.60 for patients in the highest quartile. However, zinc supplementation resulted in poorer survival. The compiled results from several studies indicate that the micronutrients that appear to be the best predicators of slower progression to AIDS include selenium and vitamins A, B12, and E [22].
The literature and our previous studies using the SIV-infected rhesus macaque model support the use of the model to examine the role that micronutrients play in the progression of SIV disease. In the present study, adding micronutrient supplementation to a diet already formulated to provide adequate levels of micronutrients had no significant effect on weight loss, BMI, fat or lean body mass, bone mineral content, or bone mineral density over time. It was also noted that the viral load set point valuethe maximum viral load achieved after infectionwas predictive of the risk of death in both groups of rhesus macaques. As would be expected, higher body weight, BMI, and height were related to better survival in both groups.
The major finding of the present study suggests that micronutrient supplementation at 23 times the estimated nutritional requirement for nonhuman primates is deleterious in SIV-infected rhesus macaques fed an otherwise healthy diet. In this model, all rhesus macaques are expected to die of their infection, and, indeed, 19 of 24 rhesus macaques became moribund during the 120 weeks of observation. Measurements of serum levels of vitamins A and E, zinc, and seleniumthe subject of a future studydid not reflect severe malabsorption or significant differences in gastrointestinal function between the groups of rhesus macaques. The high level of micronutrient supplementation, however, was associated with decreased survival time, compared with that in the nonsupplemented group, and, thus, the risk of death was increased 2.4-fold (figure 1). High levels of vitamin A were included in the supplemented diet. We did not observe either acute effects or any changes that would indicate vitamin A toxicity in the livers of these rhesus macaques at necropsy [28]. Vitamin A is an established differentiating agent. Its metabolite transretinoic acid is a potent differentiator of myeloid and lymphoid cells. By analogy, it has been shown that the differentiation of dendritic cells is associated with the increased expression of the chemokine receptor CCR5 that, in turn, increases susceptibility to HIV-1 infection [29]. The results of 2 studies [30, 31] suggest a possible role for transretinoic acid in HIV potentiation. In the first study [30], it was shown that HIV-1, which does not infect immature human myeloid cells in vitro, was capable of infecting these cells after they were treated with retinoic acid. The authors concluded that precursor cell differentiation is required for the optimal replication of HIV-1 in vitro. In the second study [31], it was demonstrated that HIV replication in cultured monocytes was 1020-fold greater when these monocytes were treated with retinoic acid for 7 days before and continuously after HIV infection.
The design of the present study was based on an initial attempt to determine if micronutrient supplementation alters the progression of disease in SIV-infected rhesus macaques. This was done to simulate supplementation in HIV-infected patients and to determine if such an intervention would be beneficial or harmful. On the basis of the present findings, it is evident that studies in which individual vitamins and minerals are added to the diet are needed to evaluate which component or components cause a more-rapid disease progression. Our data suggest that there is a need for caution in the use of megavitamin supplementation in HIV-infected patients. Although some additional micronutrients may be beneficial, it is possible that large doses are associated with adverse clinical outcomes.
Acknowledgments
We gratefully acknowledge the assistance of staff at the New England Regional Primate Research Center: Katherine George, who helped with the immunophenotyping, and the veterinary staff, who provided outstanding care to the rhesus macaques.
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